Leukocyte integrins play key roles in vascular cell adhesion in host defense, inflammation, hemostasis, and metastasis. In the last five years we have developed a set of novel tools that exploit flow cytometry to analyze cell adhesive interactions. We used flow cytometry: 1) for the real-time analysis of ligand association and dissociation with VLA-4: 2) to measure the number of bonds holding cells together under no shear conditions; 3) to measure mechanotransduction (shear-induced signaling and shear-induced bond strengthening); 4) to measure molecular conformation (adhesion molecule length) of both VLA-4 and LFA-1 in real-time on living cells by fluorescence energy transfer. The results suggest a novel mechanism of adhesion bond strengthening in which the applied force contributes to VLA-4 extension, increasing affinity and avidity, allowing VLA-4 to contribute both to low avidity leukocyte rolling and high avidity firm adhesion. Our overall goal is to define relationships among conformation, affinity, applied force and adhesive function. First, we will fill critical gaps in our understanding of VLA-4 conformational regulation in the absence of shear. We will distinguish conformational changes induced by a ligand binding event from those induced by other means of cell activation such as inside-out signaling. Second, we will compare VLA-4 (a variable capture receptor) to LFA-1 (a firm attachment receptor) which works in concert with VLA-4. Integral to this comparison is an understanding of structure-activity differences between I domain and non-l domain containing integrins. Third, we will evaluate the significance of affinity and conformation to cell adhesion. We have shown that incomplete signaling initiated by PKC and PLC differentiates between the affinity and conformational changes. We will test the idea that conformation regulates the efficiency of cell adhesion, whereas affinity regulates it duration. Fourth, we will distinguish the contributions of force (outside-in) to the duration of integrin adhesion, using a new device integrating a cuvette viscometer and a flow cytometer. By relating adhesive duration in flow cytometry to single molecule bond behavior with Evans' bioforce probe we will determine the role of integrin conformation in the adhesive process. Taken together, the results will interpret adhesion molecule behavior in terms of molecular conformation, affinity, and applied shear, in a single-cell single-molecule analysis mode and with nanoscale resolution. [unreadable] [unreadable] [unreadable]